Deregulation and Mislocalization of the Cytokinesis Regulator ECT2Activate the Rho Signaling Pathways Leading toMalignant Transformation*

Received for publication, June 25, 2003, and in revised form, November 17, 2003Published, JBC Papers in Press, November 25, 2003, DOI 10.1074/jbc.M306725200

Shin’ichi Saito‡, Xiu-Fen Liu, Keiju Kamijo, Razi Raziuddin, Takashi Tatsumoto§¶,Isamu Okamoto¶, Xiaoyan Chen, Chong-Chou Lee, Matthew V. Lorenzi�, Naoya Ohara**,and Toru Miki‡‡

From the Molecular Tumor Biology Section, Basic Research Laboratory, NCI, National Institutes of Health,Bethesda, Maryland 20892-4255

The human ECT2 protooncogene encodes a guaninenucleotide exchange factor for the Rho GTPases andregulates cytokinesis. Although the oncogenic form ofECT2 contains an N-terminal truncation, it is not clearhow the structural abnormality of ECT2 causes malig-nant transformation. Here we show that both the re-moval of the negative regulatory domain and alterationof subcellular localization are required to induce theoncogenic activity of ECT2. The transforming activity ofoncogenic ECT2 was strongly inhibited by dominantnegative Rho GTPases, suggesting the involvement ofRho GTPases in ECT2 transformation. Although dele-tion of the N-terminal cell cycle regulator-related do-main (N) of ECT2 did not activate its transforming ac-tivity, removal of the small central domain (S), whichcontains two nuclear localization signals (NLSs), sig-nificantly induced the activity. The ECT2 N domaininteracted with the catalytic domain and significantlyinhibited the focus formation by oncogenic ECT2. Inter-estingly, the introduction of the NLS mutations in the Sdomain of N-terminally truncated ECT2 dramaticallyinduced the transforming activity of this otherwise non-oncogenic derivative. Among the known Rho GTPasesexpressed in NIH 3T3 cells, RhoA was predominantlyactivated by oncogenic ECT2 in vivo. Therefore, the mis-localization of structurally altered ECT2 might causethe untimely activation of cytoplasmic Rho GTPasesleading to the malignant transformation.

The ECT2 oncogene has been isolated in a search for mito-genic signal transducers in epithelial cells, where a murine

keratinocyte expression cDNA library was introduced into fi-broblasts to induce foci of morphologically transformed cells(1). The ECT2 transfectants exhibit anchorage-independentcell growth and efficient tumor formation in nude mice. Thetransforming ECT2 cDNA encodes the C-terminal half of thefull-length protein containing Dbl-homology (DH)1 and pleck-strin homology (PH) domains, which are now found in a num-ber of molecules involved in regulation of the Rho familyGTPases. The N-terminal half of ECT2 contains domains re-lated to cell cycle control and repair proteins, including Clb6and Rad4/Cut5 (2, 3). CLB6 encodes a B-type cyclin of thebudding yeast, which promotes the transition from G1 into Sphase (4). Fission yeast cut5, which is identical to the repairgene rad4, is required for both the onset of S phase and therestraint of M phase before the completion of S phase (5). TheCut5-related domain of ECT2 consists of two repeats (6, 7),designated BRCT (BRCA1 C-terminal) repeats, which arewidespread in a number of cell-cycle checkpoint control andDNA repair proteins (7). These cell-cycle regulator-relateddomains of ECT2 play essential roles on the regulation ofcytokinesis (2, 3).

ECT2 catalyzes guanine nucleotide exchange in vitro onthree representative Rho GTPases; RhoA, Rac1, and Cdc42 (2).The Rho family of small GTPases function as molecularswitches of diverse biological functions, including cytoplasmicactin reorganization, cell motility, and cell scattering (8). Acti-vation of the Rho proteins is promoted by guanine nucleotideexchange factors, which catalyze the replacement of boundGDP by GTP. The GTP-bound form of Rho proteins can specif-ically interact with their effectors or targets and transmit sig-nals to downstream molecules. Rho proteins are inactivatedthrough the hydrolysis of bound GTP to GDP by the intrinsicGTPase activity assisted by GTPase-activating proteins(GAPs). RhoA, Rac1, and Cdc42 induce the formation of actinstress fibers, lamellipodia, and filopodia, respectively (9).

Among the known guanine nucleotide exchange factors forRho GTPases, ECT2 shows several unique characteristics.ECT2 expression is induced in S phase and reaches the highest

* This work was supported in part by a Breast Cancer Think TankAward from National Institutes of Health (NIH). The costs of publica-tion of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¶ Supported by Japan Society of Promotion of Sciences fellowships forBiomedical and Behavioral Researchers in NIH.

‡ Present address: Laboratory of Cell Biology, NCI, NIH, 37 ConventDr. MSC 4255, Bethesda, MD 20892-4255.

§ Present address: Fukuoka Teishin Hospital, 2-6-11 Yakuin, Chuo-ku, Fukuoka 810-8798, Japan.

� Present address: Oncology Drug Discovery, Pharmaceutical Re-search Institute Bristol-Myers Squibb, P. O. Box 4000, Princeton,NJ 08543-4000.

** Present address: Dept. of Bacteriology, Faculty of Dentistry, Na-gasaki University, Nagasaki 852-8588, Japan.

‡‡ To whom correspondence should be addressed: Molecular TumorBiology Section, Basic Research Laboratory, NCI, NIH, 37 Convent Dr.MSC 4255, Bethesda, MD 20892-4255. Tel.: 301-496-2289; Fax: 301-480-2512; E-mail: toru@helix.nih.gov.

1 The abbreviations used are: DH, Dbl homology; AP-1, activatorprotein-1; BRCT, BRCA1 C-terminal; CLB6, cyclin B6; Cut5, cells un-timely torn 5; DAPI, 4�, 6-diamidino-2�-phenylindole; ECT2, epithelialcell transforming gene 2 (human); ect2, mouse ECT2; Erk, extracellularsignal regulated kinase; GAP, GTPase activating protein; GFP, greenfluorescent protein; GST, glutathione S-transferase; JNK, c-jun N-ter-minal kinase; MAPK, mitogen-activated protein kinase; NLS, nuclearlocalizing signal; PH, pleckstrin homology; RFP, red fluorescent pro-tein; SRE, serum response element; SRF, serum response factor; ffu,focus-forming units; DN, dominant negative; WT, wild type.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 8, Issue of February 20, pp. 7169–7179, 2004Printed in U.S.A.

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level in G2 and M phases in regenerating mouse liver (10).ECT2 protein is specifically phosphorylated in G2 and Mphases (2). ECT2 exhibits nuclear localization in interphase,disperses throughout the cytoplasm in prometaphase, and iscondensed in the midbody during cytokinesis. Expression of adominant negative ECT2 or microinjection of anti-ECT2 anti-body strongly inhibits cytokinesis, indicating that ECT2 is acritical regulator of cytokinesis (2). Furthermore, the Drosoph-ila pbl gene, whose mutation results in the inhibition of cyto-kinesis in mitotic cycle 14 during embryogenesis, was found toencode the fly homologue of human ECT2 (11).

Although the transforming activity of several DBL familyoncogenes is stimulated by N-terminal alterations (12–15), theactivation mechanisms are still obscure. Because Rho GTPasesplay a critical role in cell transformation (16–18), ECT2 maydisplay its transforming activity through the activation of Rhoproteins. However, ECT2 is predominantly expressed in thenucleus where no expression of Rho GTPases is reported. In thepresent study, we examined the activation mechanism of thetransforming activity of ECT2. We identified the small centraldomain containing two tandem nuclear localization signals as anegative regulator of the transforming activity. We show thatelimination of these signals and a negative regulatory domainfrom ECT2 resulted in the activation of Rho GTPases in thecytoplasm, leading to malignant transformation of the cells.

EXPERIMENTAL PROCEDURES

DNA Constructs—Full-length and N-terminally truncated ECT2cDNAs were amplified by PCR using ECT2 clone 1M (2, 3) as templateand subcloned between the BamHI and EcoRI sites of the mammalianexpression vector pCEV29 or its derivative pCEV29F3, which containsthree tandem copies of FLAG sequence (19). ECT2 N-terminal deriva-tives, ECT2-N1 (amino acids 1–421), ECT2-N2 (amino acids 1–378),ECT2-N3 (amino acids 1–360), and ECT2-N4 (amino acids 1–333) havebeen described previously (3). ECT2-�S mutant lacking the S domain(amino acids 329–420) was created from two PCR products using thesame template and primers I–IV with the following sequences: I, 5�-CTC GGA TCC ATG GCT GAA AAT AGT GTA TTA-3�; II, 5�-CAG ACTCGC GGA GTA TTT GCC TTT TCA TA-3�; III, 5�-TCA CTC CGC GGTGGC AAG TTG CAA AAG AG-3�; and IV, 5�-ACT GAA TTC GGT AACGCT TCA TAT CAA ATG-3�. The PCR products synthesized usingprimer pairs I and II were digested with BamHI and BstUI. The PCRproducts generated by primers III and IV were digested with BstUI andEcoRI. These products were ligated together with the pCEV29 orpCEV29F3 vector, which had been digested with BamHI and EcoRI, tocreate ECT2-�S. Two ECT2 mutants, S1 and S2, containing RRR toAAA and R to A mutations in the NLS sequence of the S domain,respectively, were generated by the similar procedure, but followingoligonucleotides were used for PCR to introduce mutations: V, 5�-CAGACT GCG GCC GCT TTG CGA TTG CTG TTA GGG GT-3�; VI, 5�-TCACTC GCG GCC GCT TTA AAA GAA ACA CTT GCT CAG-3�; VII,5�-TTT GGC GCG CCC GGG GTG GAA ATG GTG ACA C-3�; and VIII,5�-TTT GGC GCG CCC ATC AGC TGA GCA TTC CCT T-3�. NotI andAscI were used to create S1 and S2, respectively, instead of BstUI. S3was created by the similar procedure, but S1 was used as a PCRtemplate instead of ECT2-F. ECT2-F, S1, S2, S3, �S, and �N5 were alsocloned into pEGFP-C1 (BD Biosciences/Clontech) to express ECT2 asgreen fluorescent protein (GFP) fusion proteins. All constructs gener-ated by the use of PCR were sequenced to ensure that no PCR mutationwas generated except the desired mutations.

An ECT2-�N5 derivative containing PVQR to AAAA mutations(amino acids 564–567) in the DH domain was generated by amplifyingtwo PCR fragments. Primers for the first fragment were as follows: aforward primer with a BamHI restriction site, 5�-CCC GGA TCC GCCACC ATG GTT CCT TCA AAG CAG TCA GCA-3�, and a reverse primerwith SfiI site, 5�-CAG ACT GGC CGC TGC GGC CCG GAT AAG AAGTTC AAC AAG-3�. Primers for the second fragment were: a forwardprimer with a SfiI site, 5�-TCA CTC GGC CGC AGC GGC CTT ACCCAG TGT TGC ATT ACT-3�, and a reverse primer with an EcoRI site,5�-ACT GAA TTC GGT AAC GCT TCA TAT CAA ATG-3�. PCR productswere then digested with the indicated restriction enzymes and simul-taneously ligated with the pCEV29F3 vector digested with BamHI andEcoRI. A new SfiI site was generated as a result of the introduction ofPVQR to AAAA mutations.

To introduce the SV40 NLS (nuc) into ECT2-�N5, ECT2-�N5 frag-ment was subcloned into BamHI site of pECFP-Nuc vector (BD Bio-sciences/Clontech), and then a DNA fragment containing the triplerepeats of SV40 large T antigen NLS sequence (GAT CCA AAA AAGAAG AGA AAG GTA GAT CCA AAA AAG AAG AGA AAG GTA GATCCA AAA AAG AAG AGA AAG GTA) and ECT2-�N5 sequence wasexcised and then cloned in pEGFP-C1 and pCEV29F3 vectors. Expressionof fusion proteins of expected sizes were confirmed by Western blotting.

ECT2 NLS sequence (amino acids 336–378) was attached to the 3�end of EGFP sequence (BD Biosciences/Clontech) by PCR and sub-cloned between BamHI and EcoRI sites of the mammalian EGFP ex-pression vector pCAGGFP (20) to create an EGFP-EGFP-ECT2 NLSfusion protein. The expression of 60-kDa ECT2 NLS-tagged tandemGFP protein was confirmed by Western blotting in U-2 OS cells.

Focus Forming Assays—NIH 3T3 cells were transfected with variousamounts (0.01–1.0 �g) of the eukaryotic expression vector pCEV29 orpCEV29F3 (19) containing ECT2 cDNAs or vector alone by the calciumphosphate transfection method. Focus formation was observed in un-selected plates �14 days after transfection and quantified after Giemsastaining. FLAG-tagged ECT2 variants (in pCEV29F3) showed slightlylower transforming activity than non-tagged versions (in pCEV29).Transforming activity was expressed as the number of foci per pico-moles of DNA (ffu/pmol). Comparative efficiency of transfection wasconfirmed by G-418-resistant colony formation in duplicated plates.Expression levels of the ECT2 variants were examined using anti-FLAG M2 antibody (Sigma, St. Louis, MO) and anti-GFP (BD Bio-sciences/Clontech) 48 h after transfection with 10:1 mixture of FLAG-tagged ECT2 expression vector and pEGFP-C1.

Transient Expression Reporter Gene Assays—The construction ofSRF-, and AP-1-luciferase reporter plasmids in pGL2Luc containing aminimal c-fos promoter (�56 to �109) has been described previously(21). The SRF binding sequence is derived from the serum responseelement of the c-fos gene and corresponds to SRE.mutL (22). Fireflyluciferase reporter and TK-Renilla luciferase control plasmids werecotransfected with each expression vector into COS cells. Total amountof DNA was adjusted by the addition of vector DNA. 36–48 h post-transfection, cells were lysed and luciferase activities were measuredusing the Dual-Luciferase Reporter Assay System (Promega, WI) ac-cording to the accompanied protocol. Firefly luciferase activity in thelysates was normalized to Renilla luciferase activity and expressed as arelative luciferase activity. No significant increase in luciferase activitywas observed following transfection of each expression vector DNA witha reporter plasmid containing only a luciferase cassette and c-fos min-imal promoter.

In Vitro Invasion Assays—Invasion assays were performed usingBiocoat Matrigel invasion chambers (BD Biosciences/Clontech) essen-tially as described in the manufacturer’s protocol. Matrigel invasionchambers (24-well) were rehydrated with Dulbecco’s modified Eagle’smedium containing 0.1% bovine serum albumin for 2 h at room tem-perature. NIH 3T3 cells transfected with ECT2 or control vectors wereseeded at 5 � 105/0.4 ml of medium containing 0.1% bovine serumalbumin into the inner well of invasion chambers. The outer chamberswere filled with 0.4 ml of medium containing 10% calf serum. Cells wereallowed to invade Matrigel matrices for 10–12 h at 37 °C in a CO2

incubator. To count cells that migrated onto the membrane at lowersurface, the cells on the upper side of the membrane were scraped offwith cotton swipe, then the inserts with membrane were stained withDiff-Quick (Dade Diagnostics). Cells on the lower side of membranewere photographed and counted.

Activation Assays for Rho GTPases—COS cells were transfected byGFP or GFP-ECT2-�N5 expression vectors together with pEF4/Myc-His (Invitrogen, CA) carrying inserts for AU5-tagged Rho GTPases.Lysates were prepared 24 h after transfection, and the GTP-boundforms of Rho GTPases were determined by pull-down assays usingGST-Rhotekin (for RhoA, RhoB, and RhoC) or GST-PAK PBD accordingto the manufacturer’s protocol (Cytoskeleton, Denver, CO).

Subcellular Localization and Cell Morphology—NIH 3T3 cells weretransfected with the GFP- or FLAG-tagged ECT2 expression vectorsusing the LipofectAMINE Plus reagent (Invitrogen, Carlsbad, CA).GFP-expressing cells were identified by green fluorescence. Actin andDNA were stained with rhodamine-conjugated phalloidin (Sigma) and4�,6�-diamidino-2-phenylindole (DAPI, Sigma), respectively, as reportedpreviously (2, 19). FLAG-tagged ECT2 derivatives (0.5 �g each) weretransfected into NIH 3T3 cells using the LipofectAMINE Plus reagent.Cells were fixed with a freshly prepared mixture of methanol:acetone(1:1) for 2 min at room temperature 40 h after transfection. Expressedproteins were visualized using anti-FLAG M2 monoclonal antibody-Cy3conjugate (Sigma) in the presence of 1 �g/ml DAPI. U-2 OS cells were

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also transiently transfected with the expression vectors for GFP-taggedECT2 mutants using FuGENE 6 reagent (Roche Applied Science) toconfirm their subcellular localization. In this case, Hoechst 33342 dye wasadded to culture medium at a final concentration of 10 �M, and cells weredirectly observed under the fluorescence microscope. Images were ac-quired using a Zeiss Axiovert microscope equipped with a Photometricsdigital camera and processed with IPLab software (Signal Analytics).

Time-lapse Video Microscopy—The RFP-ECT2-�N5 expression vec-tor was constructed by inserting ECT2-�N5 into pRsRed2-C1 (BO Bio-sciences/Clontech) at the BglII and EcoRI sites. pEGFP-actin was ob-tained from Clontech. NIH 3T3 cells were transfected with the equalmixture of both the plasmids using FuGENE 6 transfection reagent(Roche Applied Science). Cells were cultured on 35-mm plates in anenvironmental chamber on a stage of Zeiss Axiovert S-100 microscopeequipped with motorized X-Y-Z stages. Images were taken at 3-minintervals by using a photometric digital camera controlled by OpenLabsoftware (Improvision, Lexington, MA).

RESULTS

Deletion of the N-terminal Half of Human ECT2 Induces CellTransformation and Invasiveness—The mouse ect2 cDNA,ect2-T, which carries an N-terminal truncation, exhibits a hightransforming activity, whereas the full-length clone does notsignificantly induce transformation (23). To test if N-terminaltruncation can also activate the transforming activity of humanECT2, we generated a FLAG epitope-tagged full-length humanECT2 and its derivative ECT2-�N5, which has a similar N-terminal deletion to mouse ect2-T. Like ect2-T, ECT2-�N5 exhib-ited a high transforming activity in NIH 3T3 cells, whereas thefull-length ECT2, ECT2-F, did not show any detectable activity(Fig. 1A). Both mouse and human ECT2 similarly induced tinyfoci of transforming cells with stellate morphology, which wasdistinct from ras- or sis-induced foci (Fig. 1, A and B).

We previously reported that ect2-T stimulates anchorage-

independent growth of NIH 3T3 cells and tumorigenicity innude mice (23). To examine additional oncogenic activities ofECT2, we established NIH 3T3 clones expressing FLAG-taggedderivatives of ECT2-F, ECT2-�N5, and ECT2-N2. ECT2-N2carries the region from the N terminus to the S domain (seeFig. 6A). Western blot analysis with anti-FLAG antibodyshowed that these stable transfectants expressed FLAG-taggedECT2-F, ECT2-N2, and ECT2-�N5 at comparable levels (Fig.1C). Upon plating, ECT2-�N5-expressing cells formed second-ary foci with stellate morphology, whereas the morphology ofECT2-F and ECT2-N2 clones was indistinguishable from thevector alone transfectants (Fig. 1B). When cultured in thepresence of 10% serum, all of the stable ECT2 clones exhibitedsimilar growth properties (data not shown). However, in me-dium containing 1% serum, vector alone, ECT2-N2 andECT2-F transfectants did not grow well and the number ofviable cells gradually decreased (Fig. 2A). In contrast, ECT2-�N5 transfectants continued to grow for at least 48 h underthese conditions, suggesting that these transfectants acquiredlow serum dependence.

To test if ECT2 can induce cell invasiveness, NIH 3T3 cellsexpressing ECT2-F, ECT2-�N5, or vector alone were placed onthe surface of an artificial basement membrane, Matrigel, andthe number of the cells that had migrated through the mem-brane was counted. Interestingly, ECT2-�N5 transfectants ex-hibited a strong invasion activity, whereas ECT2-F or the vec-tor alone transfectants did not show significant activity (Fig.2B). Additionally, mouse ect2-T, which corresponds to humanECT2-�N5, also exhibited a high activity of cell invasiveness.These results indicate that oncogenic ECT2 is an efficient ac-tivator of cell invasiveness.

FIG. 1. Induction of foci of trans-forming cells by ECT2 derivatives. A,morphology of foci induced by ECT2cDNA. 1 �g each of vector alone (pCEV29)or the expression vector for human full-length ECT2 (ECT2-F), human ECT2lacking the N-terminal half (ECT2-�N5),mouse ect2 lacking the N-terminal half(ect2-T), H-ras-V12 or c-sis was used totransfect NIH3T3 cells. Cells werestained by Giemsa and then photo-graphed. B, cell morphology of stableECT2-F, N, �N5, and vector alone trans-fectants. C, expression of FLAG-taggedECT2-F, N, and �N5 in stable transfec-tants. FLAG-ECT2 fusion proteins weredetected by immunoblotting using anti-FLAG M2 monoclonal antibody.

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Oncogenic ECT2 Activates Rho Signaling Pathways—Be-cause Rho GTPases are known to regulate the JNK and p38MAPK pathways (24, 25), we examined whether these path-ways are activated in ECT2 transfectants. We first analyzedendogenous JNK activity in these ECT2 transfectants using anantibody specific to the activated form of c-Jun, which is phos-phorylated at serine 63. In vector alone transfectants, a verylow level of JNK activity was observed (Fig. 3A, top). Theactivity of JNK in these cells was increased by the stimulationwith sorbitol, an activator of the JNK pathway. The activity ofJNK was also elevated in cells expressing ECT2-�N5. In con-trast, ECT2-F or ECT2-N2 expression did not significantlyaffect the JNK activity. The JNK activity was induced bysorbitol to a similar level in all the transfectants (data notshown), indicating that all of the transfectants maintained theability to induce JNK activity. In contrast, we did not observesignificant activation of p38 or Erk MAPKs by ECT2 and itsderivatives (Fig. 3A, middle and bottom). These results indicatethat ECT2-�N5 preferentially activates the JNK signalingpathway.

Rho proteins can stimulate the transcriptional activity reg-ulated by serum response factor (SRF) (22). To examinewhether ECT2 can stimulate SRF-regulated transcription, aserum response element (SRE)-luciferase plasmid was used asa reporter. Upon coexpression of the reporter plasmid witheither the full-length or truncated ECT2 expression vector,luciferase activity was estimated. As shown in Fig. 3B, upperpanel, expression of ECT2-�N5 potently induced the transcrip-tional activity of the SRE reporter plasmid. In contrast, expres-sion of ECT2-F exhibited the activity slightly higher than thevector alone control. The SRE-regulated transcriptional activ-

ity induced by ECT2-�N5 was efficiently inhibited by either ofdominant negative RhoA, Rac1, or Cdc42. Moreover, either ofconstitutively active RhoA, Rac1, or Cdc42 efficiently enhancedSRE-mediated transcription in this system. These results sug-gest that ECT2-�N5 can stimulate SRE-mediated transcrip-tion through the activation of Rho GTPases.

We previously showed that Ost, a guanine nucleotide ex-change factor for RhoA and Cdc42, activates the transcrip-tional activity regulated by activator protein-1 (AP-1) (19). Toexamine whether ECT2 can also stimulate AP-1-regulatedtranscriptional activity, an AP-1-binding site-luciferase plas-mid was utilized as a reporter. As shown in Fig. 3B, lowerpanel, expression of ECT2-�N5 moderately elevated AP-1-reg-ulated transcriptional activity, whereas ECT2-F or ECT2-N2failed to stimulate the activity. Coexpression of dominant neg-ative RhoA, Rac1, or Cdc42 reduced the ECT2-�N5-mediatedstimulation of AP-1-regulated transcription, albeit at lowerlevels as compared with their effects on SRE-regulated tran-scription. We also found that constitutively active RhoA, Rac1,or Cdc42 efficiently stimulated AP-1-mediated transcription.Among these GTPases, Rac1 displayed the highest level ofstimulation of AP-1-regulated transcriptional activity. All ofthese results indicate that ECT2 can regulate the transcrip-tional events mediated by SRE and AP-1 through the activa-tion of Rho GTPases.

Oncogenic ECT2 Induces Cell Rounding in NIH 3T3 Fibro-blasts—Rho family proteins are involved in the organization ofactin-based cytoskeletal structures. In fibroblasts, RhoA acti-vates actin stress fiber formation, whereas Rac1 and Cdc42induce lamellipodia and filopodia formation, respectively (8).To test which actin-based structures ECT2 can induce, we

FIG. 2. Characterization of NIH 3T3cells transfected with ECT2 variants.A, growth of ECT2 transfectants in lowserum conditions. Cells were cultured inDulbecco’s modified Eagle’s medium con-taining 1% serum, and viable cells werescored at the indicated time points. B,induction of cell invasiveness by ECT2.Stable NIH 3T3 transfectants expressingthe indicated cDNA were used for Matri-gel assays to estimate cell invasiveness invitro. The cells that invaded the Matrigelswere stained and photographed (left half).The number of cells passed through theMatrigel is summarized (right half).ect2-T is a mouse cDNA with an N-termi-nal deletion similar to human ECT2-�N5.

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transiently expressed GFP-tagged ECT2-F, ECT2-�N5, or GFPvector alone in NIH 3T3 cells. A population (�20%) of cellsexpressing GFP-ECT2-�N5 showed a flat phenotype with mod-erately enhanced actin stress fiber formation (Fig. 4A, GFP-ECT2-�N5, left panels), suggesting that Rho was preferentiallyactivated by ECT2-�N5 in these cells. However, the majority ofNIH 3T3 cells expressing GFP-ECT2-�N5 exhibited a com-pacted structure with saturated F-actin staining (Fig. 4A, GFP-ECT2-�N5, right panels). In contrast, the morphology of thesurrounding cells, which were not expressing the GFP fusionprotein, was similar to that of vector alone transfectants. Thesefindings are consistent with the previous results that oncogenicECT2 induces foci containing both fusiforms and roundedcells (23).

To examine how these compacted cells were generated byECT2-�N5 expression, NIH 3T3 cells were transfected with redfluorescent protein (RFP)-fused ECT2-�N5 and GFP-fused ac-tin expression vectors. Cells expressing RFP-ECT2-�N5 wereidentified with red fluorescence, and their morphologies wereexamined by time-lapse video microscopy. In most of the cellsexpressing RFP-ECT2-�N5, actin stress fibers were disrupted27–32 h after transfection, and the cells rounded up like the Mphase cells (Fig. 4B, see the cell shown by an arrow), but mostof them did not divide in a next few hours. The cell that oncerounded nearly completely appeared to flatten to some extent(30–32 h panels) and then rounded again (33–35 h after trans-fection panels). RFP-ECT2-�N5 was detected in the entire cellof these transfectants (data not shown). In Fig. 4B, another cellexpressing ECT2-�N5 at similar level as determined by the redfluorescence also exhibited a similar morphology with addi-tional cortical activities (lower right corner). In contrast, cellsexpressing ECT2-�N5 weakly did not round up, although theyexpressed GFP-actin at a similar level (see the rightmost cell inFig. 4B). These results suggest that oncogenic ECT2 stimulates

cellular transformation by dramatically changing their actincytoskeletal morphology in NIH 3T3 cells.

ECT2 Transformation Is Dependent on the Activation of RhoGTPases—ECT2 can activate RhoA, Rac1, and Cdc42 throughguanine nucleotide exchange in vitro (2). Rho GTPases havebeen shown to play a critical role in cellular transformation(16–18). To test whether ECT2 transformation involves Rhoactivation, we cotransfected NIH 3T3 cells with the ECT2-�N5expression vector (0.1 �g) and each of the expression vectorsencoding the dominant negative (DN) forms of Rho proteins(0.5 �g). Because the addition of a large amount of DNA usuallyinhibits focus formation presumably due to the competition forthe available transcription and translation machinery in thecells, we compared the effects of wild type (WT) and DN con-structs whose difference is in a single amino acid residue. Asshown in Fig. 5A, either of dominant negative RhoA, Rac1, orCdc42 more strongly inhibited ECT2-mediated focus formationin NIH 3T3 cells than the WT counterparts. Inhibition of ECT2transformation by the empty vector was at the similar level bythe WT Rho GTPase expression vectors, and a lower amount ofWT Rho GTPases did not exhibit the inhibitory effect on ECT2transformation (data not shown). These results suggest thatECT2-�N5 induces malignant transformation through the ac-tivation of Rho GTPases.

The S Domain of ECT2 Plays a Critical Role in the Regula-tion of Transformation—To determine which domains in theN-terminal half of ECT2 regulate the transforming activity, wegenerated a set of overlapping N-terminal truncation mutants,and expressed these constructs in NIH 3T3 cells as FLAG-tagged proteins. Unexpectedly, the N-terminal deletions ex-tending to the N-terminal most region (N), CLB6-homologydomain, or each of the two BRCT domains did not activate thetransforming activity of ECT2 (Fig. 6A). However, two ECT2derivatives containing the N-terminal deletions extended to

FIG. 3. Stimulation of MAPK activ-ity and AP-1 or SRF-regulated tran-scriptional activation by ECT2. A, ef-fects of ECT2 derivative expression onMAPK activity in NIH 3T3 cells. MAPKactivity in the stable transfectants ex-pressing indicated plasmids (see Fig. 1C)was measured using phosphospecific an-tibodies, and endogenous proteins weredetected by specific antibodies (Cell Sig-naling Technology, Beverly, MA) accord-ing to the accompanied protocols. JNK ac-tivation was detected as phosphorylatedc-Jun at Ser-63, and activated Erk andp38 proteins were detected as their phos-phorylated forms. B, effects of ECT2 de-rivative expression on AP-1- or SRF-reg-ulated transcription. COS cells werecotransfected with the indicated ECT2 ex-pression vector and SRE-LUC (upper) orAP-1-LUC (lower) reporter plasmid. Acti-vation of AP-1- or SRF-regulated tran-scriptional activity was estimated by theluciferase activity. pRK-TK-Renilla lucif-erase vector was used to cotransfect thecells together with the reporter plasmidfor an internal control to normalize thetransfection/expression efficiency.

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the small central region, designated the S domain, exhibited amarkedly high transforming activity (Fig. 6A; see �N9 and�N5). To determine if the S domain itself regulates the trans-forming activity, a derivative of ECT2-F lacking the S domainwas generated. This mutant (�S) exhibited a high level oftransforming activity in NIH 3T3 cells, suggesting that the Sdomain plays a critical role in the regulation of the transform-ing activity of ECT2.

When the N-terminal deletions extended to the neighboringDH domain, the mutant ECT2 did not show detectable trans-forming activity (Fig. 6A, �N6 and �N8), suggesting that theDH domain is required for the transforming activity of ECT2.To confirm this, point mutations (PVQR3 AAAA, amino acids564–567) were introduced into the most conserved region of theDH domain in ECT2-�N5. This mutant plasmid, ECT2-�N5DH�, did not exhibit any detectable transforming activity (Fig.6A). Therefore, the DH domain, and in turn the exchangeactivity for the Rho GTPases, appeared to be essential for thetransforming activity of ECT2. We also generated a series ofC-terminal deletion mutants of ECT2-�N5. A deletion ex-tended to the PH domain (�C1) eliminated the transformingactivity from ECT2-�N5, suggesting that the PH domain is alsoessential for the transforming activity of ECT2. A small C-terminal deletion (�C3) did not affect the transforming activityof ECT2-�N5. A mutant protein lacking most of the C-terminaldomain (�C2) was weakly transforming, but the morphology ofthe foci was less aggressive than the foci induced by ECT2-�N5. The expression level of the exogenous ECT2 proteins intransiently transfected NIH 3T3 cells was examined by immu-noblotting using anti-FLAG monoclonal antibody (Fig. 6B). Allof the ECT2 derivatives were detected as proteins of expectedsizes. The expression level of all the ECT2 derivatives wascomparable with an exception of ECT2-�N5�C2, which showedmarkedly high expression. The reason for this high expressionis not known, but this expression level might contribute to thetransforming activity of ECT2-�N5�C2.

Transforming ECT2 Derivatives Partially Localize in the Cy-toplasm—We have found that ECT2 is localized in the nucleusin interphase cells (2). Several putative nuclear localizationsignals (NLSs) were found in the predicted ECT2 sequence,including RKRRR (amino acids 346–350) and PRKRP (369–373) located in the S domain (Fig. 6A). Like endogenous ECT2,exogenously expressed FLAG-ECT2-F also localized in the nu-cleus, whereas FLAG-ECT2-�N5, which lacks both the NLSs,was detected in both the nucleus and cytoplasm (Fig. 6C).GFP-tagged ECT2 derivatives also showed similar localizationpatterns to their FLAG-tagged counterparts (data not shown).ECT2-�N5 contains a putative NLS at the C-terminal domain.GFP-ECT2-C, which consisted of the C-terminal domain alone(amino acids 753–882), was localized predominantly in thenucleus (data not shown), suggesting that the NLS in theC-terminal domain is functional. Like ECT2-�N5, ECT2-�N9,which lacks the N-terminal NLS, had a high transformingactivity, whereas ECT2-�N4, which contains both the NLSs inthe S domain, did not exhibit detectable transforming activity(Fig. 6A). ECT2-�N9 also exhibited both the nuclear and cyto-plasmic localization (data not shown). Therefore, the trans-forming activity of ECT2 derivatives coincided with partialnuclear localization.

Loss of Nuclear Localization Signals Affects the Transform-ing Activity of ECT2—To test whether the NLS sequenceslocated in the S domain are responsible for the activation of thetransforming activity of ECT2, we mutated the first (N-termi-nal) NLS (RKRRRLK) to RKAAALK in ECT2-F. Because NLSsare usually rich in the positively charged amino acids Lys andArg, Arg to Ala changes in NLSs should reduce the nuclearlocalization of the protein. This mutant, ECT2-S1, exhibited asignificant transforming activity, whereas ECT2-F did nothave any detectable activity (Fig. 6A). ECT2-S2, an ECT2-Fderivative containing a mutation in the second NLS also ex-hibited a weak transforming activity. We also generated ECT2-S3, which contains mutations in both the NLSs in the S do-main. This mutant also showed a significant transformingactivity in NIH 3T3 cells. Subcellular localization analysis re-

FIG. 4. Induction of cytoskeletal actin remodeling by onco-genic ECT2. A, actin organization of ECT2 transfectants. NIH 3T3were transfected with the GFP-ECT2-�N5 expression vector or GFPvector. Cells expressing GFP-ECT2-�N5 or GFP (indicated by arrows)were identified by green fluorescence and stained with rhodamine-conjugated phalloidin for F-actin. B, time-lapse video recording of themorphological changes of NIH 3T3 cells upon oncogenic ECT2 transfec-tion. NIH 3T3 fibroblasts were transiently transfected with GFP-actinand RFP-ECT2-�N5, and cells with green fluorescence were photo-graphed using time-lapse video microscopy. An arrow indicates one ofthe RFP-ECT2-�N5-expressing cells, which showed morphologicalchanges. The number in each panel indicates time after transfection (h).Bar, 10 �M.

FIG. 5. Inhibition of ECT2-�N5-induced transformation bydominant negative Rho GTPases. NIH 3T3 cells were cotransfectedwith ECT2-�N5 and indicated Rho expression vectors. Foci of morpho-logically transformed cells were scored 14 days after transfection andexpressed as percentages of the number of foci induced by ECT2-�N5alone. Similar transfection efficiency of NIH 3T3 cells with the vectorsused was confirmed by the formation of G418-resistant colonies. Shownis one of the results reproduced three times with an identical pattern.

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vealed that ECT2-S3 localized in the cytoplasm as well as inthe nucleus (Fig. 6C). ECT2-S1 and -S2 also exhibited a similarlocalization to ECT2-S3 (data not shown). The expression levelsof the wild type and mutant proteins were comparable (Fig.6B). These results suggest that the two NLSs in the S domain

can function in vivo and that the impairment of NLSs canactivate the transforming activity of ECT2.

To further verify that ECT2 NLSs can function as nuclearlocalization signals, these NLSs were introduced into tandemGFP. Whereas GFP exhibited both the cytoplasmic and nuclear

FIG. 6. Mapping of the domains that regulate transforming activity of ECT2. A, deletion mapping of domains that affect transformingactivity. Regions carried by the ECT2 derivatives are shown by horizontal bars under a schematic representation of the human ECT2 protein.BRCT-1 and BRCT-2 indicate BRCA1 C-terminal repeats (7). The numbers at the ends of each clone represent the amino acid numbers relativeto ECT2-F. �S contains an internal deletion of the S domain. �N5 DH� contains PVQR to AAAA substitutions at amino acids 564–567, whoselocation is also indicated by x. Transforming activity was shown as follows: �, �1 � 100 ffu/pmol; �. 1 � 100 � 1 � 101 ffu/pmol; ��, 1 � 103 �1 � 104 ffu/pmol; and ���, � 1 � 104 ffu/pmol. B, identification of exogenously expressed FLAG-ECT2 fusion proteins by immunoblotting. NIH3T3 cells were transiently transfected with indicated FLAG-tagged ECT2 derivatives together with the GFP expression vector pEGFP-C1.Forty-eight hours after transfection, cells were lysed and the proteins expressed from the vectors were analyzed by immunoblotting withanti-FLAG (M2), anti-GFP, and anti-� tubulin antibodies. The GFP expression level was measured to monitor the transfection efficiency of eachECT2 variant and to detect the possible effect of ECT2 derivatives on protein expression. �-Tubulin expression was measured as a loading control.Bands with expected sizes are indicated by dots at the right side. C, subcellular localization of the FLAG-ECT2 fusion proteins. The fusion proteins(red) and nuclei (blue) were detected by anti-FLAG antibody and DAPI, respectively. Merged images are shown at the bottom.

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localization due to its relatively small size, tandem GFPshowed reduced nuclear localization (Fig. 7A). In contrast, thederivative of tandem GFP containing ECT2 NLSs displayedpredominant nuclear localization, indicating that ECT2 NLSsare functional nuclear localization signals.

To test whether cytoplasmic localization of ECT2-�N5 can bereduced by the addition of another nuclear localization signal,we introduced SV40 nuclear localization signal (nuc) intoECT2-�N5. As shown in Fig. 7B, ECT2-�N5 nuc displayedpredominant nuclear localization, whereas ECT2-�N5 exhib-ited both the cytoplasmic and nuclear localization. Addition-ally, GFP-tagged ECT2-�N5 displayed a relatively weak butsignificant transforming activity in NIH 3T3 cells, whereasGFP- ECT2-�N5 nuc did not show detectable transformingactivity (data not shown). All of these results strongly suggestthat cytoplasmic localization of ECT2 is required for its trans-forming activity.

The N-terminal Domain of ECT2 Interacts with the CatalyticDomain—Although the N-terminal truncations that did notextend to the S domain did not activate the transforming ac-tivity of ECT2, the introduction of NLS mutations to ECT2-F

stimulated its transforming activity. However, the level of ac-tivation by NLS mutations was very low compared with thedeletion of the N-terminal half (�N5). The deletion of the entireS domain (�S) markedly stimulated the transforming activity,but the transforming activity of ECT2-�S was still lower thanECT2-�N5, suggesting that the sequence of the S domain otherthan the NLSs also negatively regulates the transforming ac-tivity. We reasoned that, although the deletions of N-terminaldomains themselves cannot induce the transforming activity ofECT2, these domains may inhibit the catalytic activity of ECT2by the interaction with the C-terminal half. To test this possi-bility, GFP-tagged ECT2-N4 was coexpressed with FLAG-tagged ECT2-F, -N4, -�N5, or -DH in COS cells. When GFP-ECT2-N4 was immunoprecipitated with anti-GFP antibody,FLAG-tagged ECT2-F, -�N5, and -DH were detected in theimmunoprecipitates (Fig. 8A). Particularly, FLAG-ECT2-DHexhibited a strong association with GFP-ECT2-N4. In contrast,FLAG-ECT2-N4 was not coimmunoprecipitated with GFP-ECT2-N4. These results suggest that the N-terminal domain ofECT2 can associate with the catalytic domain.

We further examined whether ECT2 N-terminal derivativespossess the capability to inhibit the transforming activity ofECT2-�N5. Because we previously found that ECT2-N4

FIG. 7. Functional comparison of ECT2 NLS and SV40 NLS. A,subcellular localization of ECT2 NLS-tagged tandem GFP protein inU-2 OS cells. U-2 OS cells were transiently transfected with the indi-cated expression vectors using FuGENE 6 reagent. Hoechst 33342 dyewas added to culture medium at a final concentration of 10 �M 24 hafter transfection, and cells were directly observed under the fluores-cence microscope and photographed. B, subcellular localization of SV40NLS (nuc)-tagged ECT2-�N5 in U-2 OS cells. U-2 OS and NIH 3T3(data not shown) cells were transiently transfected with the indicatedexpression vectors using FuGENE 6 and LipofectAMINE PLUS rea-gent, respectively. Hoechst 33342 dye was added to culture medium ata final concentration of 10 �M 24 h after transfection, and cells weredirectly observed under the fluorescence microscope and photographed.

FIG. 8. Interaction of the N- and C-terminal domains of ECT2.A, association of ECT2-N with ECT2-�N5 or ECT2-DH. Lysates pre-pared from COS cells expressing indicated proteins were subjected toimmunoprecipitation by anti-GFP or anti-FLAG antibodies, and blottedby anti-FLAG antibody. *, location of immunoglobulin heavy chain. V,FLAG vector; F, FLAG-ECT2-F; N4, FLAG-ECT2-N4; �N, FLAG-ECT2-�N5; DH, FLAG-ECT2-DH (amino acids 414–637). B, inhibitionof ECT2-�N5-transforming activity in NIH 3T3 cells by ECT2-N. NIH3T3 cells were transfected with ECT2-�N5 together with the indicatedexpression vectors in triplicate, and foci of transforming cells werescored 14 days after transfection. The number of foci induced by ECT2-�N5 plus vector alone was normalized to 100%. Similar experimentswere performed using H-rasV12 as control. Shown are representativeresults reproduced twice with an identical pattern.

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strongly inhibits cytokinesis (2), we used ECT2-N1, which con-tains the entire S domain, and ECT2-N3, which contains onlythe N-terminal NLS in the S domain, for this analysis. Asshown in Fig. 8B, either of ECT2-N1 or -N3 significantly inhib-ited the transforming activity of ECT2-�N5. In contrast, eitherECT2-N1 or -N3 did not significantly affect the transformingactivity of the H-ras oncogene.

Transforming Activity of N-terminally Truncated ECT2 IsStrongly Stimulated by the Loss of Nuclear Localization Sig-nals—As ECT2-N can associate with the catalytic domain, suchan intramolecular association may inhibit the catalytic activityof ECT2. This raised the possibility that the loss of NLSs fromECT2-�N4, which lacks the N-terminal domains but retainsthe S domain, activates the transforming activity. To explorethis possibility, the NLS mutations were introduced into the Sdomain of ECT2-�N4, and their transforming activity was de-termined (Fig. 9). Whereas ECT2-�N4 did not exhibit anydetectable activity, ECT2-�N4S1, which contains mutations inthe first NLS showed a strong focus formation in NIH 3T3 cells.The introduction of S3 mutations (S1�S2) to ECT2-�N4 alsoexhibited a strong transforming activity. The transforming ac-tivity of ECT2-�N4S1 and ECT2-�N4S3 was comparable tothat of ECT2-�S. As ECT2-�N5, which lacks the entire Sdomain, showed a higher activity, the NLS mutations mightnot strong enough to fully activate the transforming activity.These results suggest that loss of NLSs strongly activates thetransforming activity when the N-terminal domain is notpresent.

RhoA Is Strongly Activated by Cytoplasmic ECT2—Theabove results suggest that the cytoplasmic localization of ECT2derivatives lacking the N-terminal cell cycle regulator-relateddomains activate cytoplasmic Rho GTPases leading to malig-nant transformation. To test which Rho GTPases are activatedin the cytoplasm by such the ECT2 derivatives in vivo, wecotransfected COS cells with expression vectors for AU5-taggedRho GTPases and GFP-tagged ECT2-�N5 or GFP alone, andGTP-bound Rho GTPases were pulled down with GST-taggedRho-binding domain of Rhotekin (GST-RBD) or p21-bindingdomain of PAK (GST-PBD). However, the initial results indi-cated that the expression level of exogenous Rho GTPases wasaffected by the ECT2 expression vector and thus the amount ofRho GTPases pulled down with GST-RBD or GST-PBD did not

reflect the exchange activity of ECT2 (data not shown). There-fore, we subcloned the AU5-Rho inserts into the EF-1� promot-er-based expression vector and transfected them together withthe ECT2-�N5 expression vector into COS cells. As shown inFig. 10A, the expression level of exogenous Rho GTPases werenot affected by ECT2-�N5 expression. RhoA was efficientlyactivated by ECT2-�N5. RhoB and RhoC were also activated byECT2-�N5 albeit less efficiently. In these experiments we usedAU5-tagged Rho GTPases to avoid possible cross reactivity ofspecific antibodies. However, the detection of activation of en-dogenous RhoA, RhoB, and RhoC by ECT2-�N5 using specificantibodies revealed similar results (data not shown). Rac2 wasalso dramatically activated by ECT2-�N5, and Rac1 was acti-vated moderately. In contrast, activation of Cdc42 and TC10was below the detectable level under these conditions (data notshown).

To test whether ECT2-N affects the exchange activity ofECT2 in vivo, we cotransfected COS cells with ECT2-�N5 andECT2-N. GTP-bound RhoA was pulled down by GST-RBD andthen detected with anti-RhoA antibody (Fig. 10B). ECT2-N4,which lacks NLSs, relatively weakly but significantly reducedthe accumulation of GTP-RhoA by ECT2-�N5. In contrast,ECT2-N2, which contains NLSs, did not affect the GTP-RhoAaccumulation. We also tested a higher amount of ECT2-NDNAs (Fig. 10B; ��), but they nonspecifically inhibited GTP-RhoA accumulation.

FIG. 9. Effects of mutations in the nuclear localization signalson the transforming activity of ECT2-�N4. NIH 3T3 cells weretransfected with the indicated plasmids, and foci of morphologicallytransformed cells were scored 14 days after transfection.

FIG. 10. Identification of Rho GTPases activated by oncogenicECT2. A, COS cells were transfected with the indicated AU5-taggedRho expression vectors together with the GFP vector (�) or GFP-ECT2-�N5 expression vector (�). The GTP-bound forms of Rho GTPases werepulled down by GST-RBD (for RhoA, RhoB, and RhoC) or GST-PBD (forRac1 and Rac2) and detected by anti-AU5 antibody. The amount ofGST-RBD and GST-PBD in the reaction mixtures was also determinedby protein staining. B, effects of ECT2-N derivatives on ECT2-�N5-induced GTP-RhoA accumulation. COS cells were transfected withECT2-�N5 (0.5 �g) and indicated ECT2 derivatives (�, 1.5 �g; ��, 3.5�g). The GTP-bound forms of Rho GTPases were analyzed as in panel A.Similar results were reproduced three times.

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DISCUSSION

In the present study, we characterized the malignant trans-formation induced by the human ECT2 protooncogene. Likemany other Dbl family proteins, ECT2 also contains the DHand PH domains. However, ECT2 is unique among these pro-teins in that it contains cell cycle control domains in its N-terminal half and nuclear localization signals in the centraland C-terminal domains. The transforming activity of ECT2can be activated by the deletion of the N-terminal half. Inaddition to the previously reported oncogenic properties, wealso found that ECT2-�N5 transfectants exhibited elevated cellinvasiveness and reduced serum dependence.

We found that oncogenic ECT2 activates several RhoGTPases-regulated signaling pathways. It has been shownthat Rac1 and Cdc42 can activate JNK and p38 MAPK cas-cades (24, 25). ECT2 efficiently activated JNK, but not p38 andErk pathways, at a detectable level. Although the differences ofexperimental conditions and sensitivity of the detection mayexplain why ECT2 did not activate p38 efficiently, ECT2 mayassociate with cellular components, which are specifically in-volved in JNK signaling. It is also possible that stimulation ofthe cycling of GDP- and GTP-bound forms of Rho GTPases byexchange factors may activate the JNK pathways more effi-ciently than p38 pathway, whereas mutationally activatedRac1 and Cdc42 can efficiently stimulate both the pathways.Because Rac2 is specifically expressed in cells of hematopoieticlineages (26), ECT2 may activate JNK through Rac1. However,we cannot rule out the possibility that ECT2 activates JNKthrough Cdc42 or other Rho family of GTPases, as theseGTPases may be activated by ECT2 under certain conditions.

We have demonstrated that activated ECT2 can induce SRE-or AP-1-regulated transcriptional activity. Although the acti-vation of SRE-regulated transcription by ECT2 was efficientlyinhibited by DN-Rho GTPases, AP-1-regulated activity wasmarginally inhibited by these mutant GTPases. This indicatesthat activation of AP-1-regulated transcription by ECT2 ispartly attributed to the activation of Rho GTPases. Therefore,ECT2 may also activate other pathways to stimulate AP-1-mediated transcription.

It has been reported that microinjection of porcine aorticendothelial cells with an oncogenic form of mouse ect2 inducedlamellipodia formation (27). We also observed a similar pheno-type in COS cells expressing ECT2-�N5 (data not shown).However, a population of NIH 3T3 fibroblasts expressingECT2-�N5 exhibited enhanced stress fiber formation. Theseresults may suggest that different Rho GTPases are activatedby ECT2-�N5 in different cell types. We also found that themajority of the NIH 3T3 cells expressing ECT2-�N5 were com-pletely rounded up and actin stress fibers appeared to havebeen disrupted. These results were consistent with the previ-ous observations that ECT2-induced foci contained bothrounded cells and fusiforms (23). Time-lapse microscopy anal-ysis revealed that the morphological change upon ECT2-�N5expression is a dynamic event, which oscillates betweenrounded and partially flatten cell shapes. It is possible thatactivation of Rho GTPases was controlled in a temporal man-ner: upon the expression of oncogenic ECT2, Rho might beactivated to induce stress fibers, but at a later stage thesestress fibers might have been disrupted by unknown mecha-nisms, which can generate rounded cells. Because untrans-formed cells also round up immediately before cell division intheir normal cell cycle, it would be of interest to investigatewhether or not the cytokinesis regulator ECT2 has an addi-tional role in the control of cell rounding.

We utilized focus formation assays to determine the regionthat regulates the oncogenic activity of ECT2. We found that

mutations in the conserved amino acids in the DH domainefficiently abolished the transforming activity of ECT2. Theseresults indicate that the activation of Rho GTPases is criticalfor cell transformation by ECT2. This conclusion is furthersupported by the finding that the transforming activity ofECT2 was efficiently inhibited by DN Rho GTPases. The DNRhoA, Rac1, and Cdc42 inhibited ECT2 transformation at asimilar level and no significant difference was observed amongthe GTPases. Because DN GTPases were thought to tightlybind their exchange factors to inhibit downstream signaling(28), these results are consistent with our previous findingsthat ECT2 activates RhoA, Rac1, and Cdc42 in vitro.

We found that deletion of cell cycle regulator-related do-mains at the N-terminal half of ECT2 alone did not activate thetransforming activity. Unexpectedly, deletion of the S domainwas a critical factor for the transforming activity. The S domaincontains two tandem nuclear localization signals and the ECT2derivatives lacking the S domain partially localized in thecytoplasm. Because Rho proteins are known to localize in thecytoplasm and membrane fractions, a spill over of ECT2 intothe cytoplasm might result in untimely Rho activation andeventually cause malignant transformation. The result thatthe ECT2 derivatives containing mutations at the NLSs in theS domain partially localized to the cytoplasm and exhibited anelevated level of the transforming activity further suggests thecytoplasmic localization of ECT2 as a major cause of the trans-forming activity. However, the activation level of the trans-forming activity of ECT2 by the NLS mutants was very weakwhen compared with that of ECT2-�N5. Therefore, cytoplasmiclocalization itself might not be sufficient to fully activate thetransforming activity of ECT2. The introduction of the NLSmutants into the N-terminally truncated ECT2, �N4, dramat-ically induced its transforming activity (Fig. 9). These resultssuggest that, although the deletion of the N-terminal cell cycle

FIG. 11. A model of ECT2 activation and malignant transfor-mation. The catalytic domain (DH) of ECT2 is inhibited by the bindingof the N-terminal domain. ECT2 cannot activate Rho GTPases, becauseit is sequestered in the nucleus in interphase cells (inactive, nuclear).An ECT2 derivative lacking the N-terminal domain, but retaining the Sdomain, may be active, but it still cannot activate the Rho GTPases,because it localizes in the nucleus (active, nuclear). An ECT2 derivativehaving the �S deletion lacks two NLSs and, therefore, becomes par-tially cytoplasmic (active, partially cytoplasmic). This allows the ECT2derivative to activate Rho GTPases in the cytoplasm and thus causesmorphological transformation of the cells (lower part).

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regulator-related domains itself cannot induce the transform-ing activity, partial cytoplasmic localization of such the ECT2derivative efficiently induces cell transformation.

Based on these results, we propose the following model onECT2 activation (Fig. 11). ECT2 is localized in the nucleus ininterphase cells (2). In this stage, ECT2 may be in an inactivestate, where the N-terminal domain interacts with the catalyticdomain to inhibit its exchange activity. The deletion of theN-terminal cell cycle domains activates the catalytic activityof ECT2. However, this molecule still cannot activate RhoGTPases in vivo, because such ECT2 derivatives still localize inthe nucleus. The removal of the S domain eliminates two NLSs,which causes partial cytoplasmic localization of such the mol-ecules. Cytoplasmic localization of N-terminally truncatedECT2 activates Rho GTPases, resulting in malignant transfor-mation (Fig. 11, lower panel). As the introduction of the NLSmutations to the N-terminally truncated ECT2 strongly acti-vated its transforming activity (Fig. 9), the loss of the NLSsappeared to be the major cause of the activation of the trans-forming activity of ECT2. Because the introduction of �S dele-tion was sufficient to induce the transforming activity of ECT2,the S domain might also be involved in the negative regulationof the catalytic activity. However, because the transformingactivity of ECT2-�S was still lower than ECT2-�N5 (Fig. 9), theremoval of the S domain might only partially remove the neg-ative regulation of the catalytic domain, and N-terminal trun-cation is required for full activation of the transformingactivity.

Examination of Rho GTPases activation in vivo using theGST-RBD and GST-PBD fusion proteins revealed that RhoA isstrongly activated by ECT2-�N5. RhoB and Rac1 were alsomoderately activated. We also observed a weak activation ofRhoC, but Cdc42 activation was under detection. Because thedifferent GST fusion proteins were used to estimate the GTP-bound forms of these GTPases, and affinities of these proteinsto these GTPases are also different, it would be difficult tocompare the activation levels of the GTPases in accuracy. It ispossible that Cdc42 activation in vivo is less efficient than invitro by some reasons. For example, cytoplasmic activation ofRho may require a scaffold protein, and ECT2 and RhoA, butnot Cdc42, may be recruited to such the scaffold. Alternatively,the exchange activity of ECT2 on Cdc42 may be stimulated bya modification such as phosphorylation. Such an example ofsubstrate specificity conversion has been shown in a recentreport that phosphorylation by Aurora B converts MgcRacGAPto a RhoGAP during cytokinesis (29). As Rac2 expression isrestricted is in the cells of hematopoietic lineages (26), andRhoC is not reportedly expressed in NIH 3T3 cells (30), RhoA,RhoB, and Rac1 might be involved in transformation by ECT2-�N5. Among them, the highest activation level of RhoA byECT2-�N5 may indicate the major role of this GTPase inECT2-mediated cell transformation.

Although the Rho family of GTPases are involved in variousbiological functions in cells and tissues, the localization of theirexchange factors is strictly restricted to certain cell typesand/or specific subcellular structures. In the case of ECT2, itslocalization to the cleavage furrow and midbody appears todetermine its role in the regulation of cytokinesis. In inter-

phase cells, ECT2 is localized to the nucleus. This subcellularlocalization might insulate ECT2 from its substrates and thusprevent malignant transformation. Additionally, ECT2 mayhave a nuclear function such as DNA damage and replicationcheckpoint control as suggested by the function of Cut5 (6, 31).ECT2 is released to the cytoplasm in prometaphase after nu-clear membrane breakdown (2), and subsequent activation ofRho GTPases is thought to trigger cytokinesis. An impedimentof such the sophisticated regulatory mechanisms might resultin mislocalization of ECT2 and untimely Rho activation leadingto malignant transformation of the cells.

Acknowledgments—We thank Drs. J. Pierce, L. Samelson, and D.Lowy for support; V. Kapoor for technical assistance; and Drs. J. S.Gutkind and S. Narumiya for materials.

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MikiOkamoto, Xiaoyan Chen, Chong-Chou Lee, Matthew V. Lorenzi, Naoya Ohara and Toru Shin'ichi Saito, Xiu-Fen Liu, Keiju Kamijo, Razi Raziuddin, Takashi Tatsumoto, Isamu

Rho Signaling Pathways Leading to Malignant TransformationDeregulation and Mislocalization of the Cytokinesis Regulator ECT2 Activate the

doi: 10.1074/jbc.M306725200 originally published online November 25, 20032004, 279:7169-7179.J. Biol. Chem. 

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